Pregnancy-related enzyme activity

ABSTRACT

This invention relates to the role of the enzyme proprotein convertase 5/6 in pregnancy, and in particular to the detection or modulation of proprotein convertase 5/6 and its isoforms in the uterus. This enzyme is useful in the control of fertility, the monitoring of early pregnancy, and for the detection of uterine receptivity. The invention also relates to methods of screening for compounds which have the ability to modulate the activity or expression of proprotein convertase 5/6, which may be useful in regulating fertility in mammals. Novel forms of proprotein convertase 5/6 are also disclosed and claimed.

This invention relates to the role of the enzyme proprotein convertase5/6 in pregnancy, and in particular to the detection or modulation ofproprotein convertase 5/6 and its isoforms in the uterus. This enzyme isuseful in the control of fertility, the monitoring of early pregnancy,and for the detection of uterine receptivity. The invention also relatesto methods of screening for compounds which have the ability to modulatethe activity or expression of proprotein convertase 5/6, which may beuseful in regulating fertility in mammals.

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art, in Australia or in any othercountry.

Embryo implantation, the process by which the blastocyst attaches to andimplants in the uterus, leads to the establishment of an intimaterelationship between the embryo and the endometrium. Implantation is oneof the most important limiting factors in establishing a successfulpregnancy. It is a complex process involving active interactions betweenthe blastocyst and the uterus. The uterus must undergo dramaticmorphological and physiological changes to transform itself from anon-receptive to a receptive state. This differentiation process isprimarily mediated by the coordinated effects of the ovarian-hormones,which act through their intracellular receptors to regulate geneexpression, and hence to influence cellular proliferation anddifferentiation.

While the details of the exact molecular events occurring in the uterusduring this differentiation process towards receptivity are stillunknown, in principle it can be predicted that a unique set of genes isup- or down-regulated in a temporally and spatially specific manner.Indeed, induction of specific genes in the uterus during theperi-implantation period, including those encoding some growth factorsand cytokines, has been reported (Huet-Hudson et al, 1990; Stewart etal, 1992; Zhu et al, 1998; Robb et al, 1998; Das et al, 1999). However,given the complexity and the as-yet imprecisely defined molecularmechanisms of the process, many other molecules critical forimplantation are still unidentified.

We have used the mouse as a model in a search for hitherto unrecognisedmolecules which are important in the early stages of implantation. Inthe mouse on day 4.5 of pregnancy, the uterus undergoes dramaticmorphological changes in association with cell proliferation anddifferentiation, leading to the acquisition of a receptive state (Finn &McLaren, 1967; Abrahamsohn & Zorn, 1993). This uterine remodelling isassociated with an increase in vascular permeability at implantationsites (Psychoyos, 1973).

We hypothesised that the proliferation and differentiation ofendometrial cells at this time is associated with up- or down-regulationof a number of genes, many of which are still unknown (Nie et al, 1997).To identify uterine genes which are potentially critical for uterinereceptivity, we used the technique of RNA differential display (DDPCR)(Liang & Pardee, 1992 & 1993), and compared the mRNA expression patternsof implantation and interimplantation sites on day 4.5 of pregnancy (Nieet al, 2000a & b).

We found that one of the genes which was differently regulated betweenthe two sites was that encoding proprotein (prohormone) convertase 5/6(PC 5/6), a member of a serine proteinase family which is responsiblefor processing of precursor proteins to their active forms by selectiveproteolysis. PC 5/6 has not been previously detected in the uterus, andno role for this enzyme during embryo implantation has been suggested.

A cDNA encoding PC5, as well as the complete amino acid and nucleotidesequence of human PC5, is disclosed in PCT/CA97/00535. PC5 is describedas being a prorenin-processing enzyme which is overexpressed inartherosclerotic coronary arteries. Inhibition of smooth muscle cellproliferation by antisense oligonucleotides directed against PC5 isdescribed, as well as the use of such oligonucleotides in the preventionof restenosis. PC5 RNA was also detected in the post-partum placentalcotyledon and in the testes.

Miranda et al. (1996) describes the presence of two isoforms of humanPC6 protease in human T cells and its role in HIV-1 gp160 processing inCD4+ T cells. Its distribution in the intestine, adrenal glands, lung,ovaries, testes, brain, spleen, kidney and liver is described. There isno mention of the presence of PC6 in the uterus.

Only the role of PC5 in the cleaving of human prorenin is discussed.There is no disclosure or suggestion in either of these documents thatPC5 might be present in the uterus, or that it might play a role infertility.

PC 5/6 has been detected in the thyroid, parathyroid, gut, adrenals,nervous system, and the cardiovascular system, but has not been reportedas being present in any gonadal tissue or other reproductive organ.(Seidah & Chretien, 1999). In another paper (Rancourt & Rancourt, 1997)there is a mention of PC6 expression detected by in situ hybridisationin decidua and trophoblast cells, but no additional data is provided.

We have examined the uterine expression of PC 5/6 during early pregnancyin the mouse, and have demonstrated for the first time that thisproprotein convertase is specifically upregulated in the mouse uterus atthe sites of embryo attachment, and that its expression is restricted tothe decidualized stromal cells. We have also identified a novel isoformof PC 5/6, which appears to be specifically expressed in implantationsites of the uterus during the embryo's implantation period. We havealso detected PC 5/6 expression in human endometrium.

SUMMARY OF THE INVENTION

Proprotein convertase 5/6 is believed to be useful in promoting theimplantation of the fertilized egg in the uterus, development of theembryo and maintenance of pregnancy. Therefore modulation of theactivity of this enzyme may be used to promote or to inhibit fertility.It is contemplated that the fertility-promoting methods of the inventionwill be particularly useful in assisted reproduction programs, includingbut not limited to in vitro fertilisation and gamete intrafallopiantransfer methodologies. It is also contemplated that modulation of PC5/6 activity could be used to prevent the establishment of a pregnancy,ie that this could be used as a contraceptive.

In a first aspect the invention provides a method of converting theuterus of a female mammal from a non-receptive to a receptive state,comprising the step of administering an effective amount of proproteinconvertase 5/6 (PC 5/6), or an agonist thereof, to a female mammal inneed of such treatment.

In a second aspect, the invention provides a method of promoting theimplantation of a fertilised egg in a host uterus, comprising the stepof administering a proprotein convertase 5/6, or an agonist thereof, toa female mammal in need of such treatment.

In a third aspect, the invention provides a method of detecting thefertile period in a female mammal, comprising the step of measuring theactivity of or detecting the presence of proprotein convertase 5/6 in abiological sample from the mammal. The biological sample may be uterinetissue, washings from the uterine cavity, or another biological fluid,such as blood, plasma, serum or saliva.

In a fourth aspect, the invention provides a method of promotingfertility of a female mammal, comprising the step of stimulating theactivity of proprotein convertase 5/6, in the uterus of a female mammalin need of such treatment. In these first, second and fourth aspects ofthe invention it will be clearly understood that the method of theinvention may be used to treat either a female mammal who is to receiveone of her own fertilised eggs, or one who is to receive a fertilisedegg from a donor female.

In a fifth aspect, the invention provides a method of detecting whetherthe uterus of a female mammal is in a receptive state, comprising thestep of detecting the presence or absence of PC 5/6, or its presence inincreased amounts at a particular stage of the cycle compared withanother stage, whereby the presence of or an increase in PC 5/6 meansthat the uterus is in a receptive state. The presence or absence of PC5/6 may be assessed by detecting the activity of the enzyme itself, eg.using a specific antibody directed against the enzyme, or by detectingnucleic acid, encoding the enzyme, for example using PCR or in in situhybridization.

In a sixth aspect, the invention provides a method of detecting an earlypregnancy, comprising the step of detecting the presence of PC 5/6 or anincrease of PC 5/6 above the level in the non-pregnant state.

In a seventh aspect, the invention provides a method of inhibiting theconversion of a non-receptive state of the uterus of a female mammal toa receptive state, comprising the step of administering an antagonist ofPC 5/6 to a female mammal in need of such treatment.

In an eighth aspect, the invention provides a method of inhibitingfertility, including but not limited to inhibiting uterine receptivityand/or embryo implantation, in a female mammal, comprising the step ofadministering an antagonist of PC 5/6 to a female mammal in need of suchtreatment.

In a ninth aspect, the invention provides a method of screening forcompounds which have the ability to modulate the activity of proproteinconvertase 5/6, comprising the step of assessing the ability of acandidate compound to increase or decrease proprotein convertase 5/6activity. The person skilled in the art will appreciate that a widerange of activities can be measured, for example stimulation of geneexpression, stimulation of activation of PC 5/6 or inhibition ofdegradation by PC 5/6.

In a tenth aspect, the invention provides a method of identifyingmolecules necessary for implantation, comprising the step of testing acandidate molecule for the ability to promote the conversion of proteinprecursors cleavable by PC 5/6 into mature proteins.

In an eleventh aspect, the invention provides a nucleic acid moleculeencoding an isoform of PC 5/6 which: (a) is present at the implantationsites in pregnant uterus during the implantation period but not inintestine or other tissues known to contain PC 5/6; (b) encodes an RNAtranscript of about 5.5 kb; and (c) is able to hybridize under at leastmoderately stringent conditions to the sequence: 1 GAAGTTAGTT GTGCGCGCTGCTTAGCGCGC GAGCCAGCGG GCGGGCGAAG SEQ ID NO: 4 51 GCGGCGAAGC GTCGGGACCATGGACTGGGA CTGGGGGAAC CGCTGCAGCC 101 GCCCGGGACG GCGGGACCTG CTGTGCGTGCTGGCACTGCT CGCCGGCTGT 151 CTGCTCCCGG TATGCCGGAC GCGCGTCTAC ACCAACCACTGGGCAGTGAA 201 GATCGCCGGC GGCTTCGCGG AGGCAGATCG CATAGCCAGC AAGTACGGAT251 TCATCAACGT AGGACAGATC GGTGCACTGA AGGACTACTA TCACTTCTAC 301CATAGTAGGA CCATTAAAAG GTCTGTTCTC TCGAGCAGAG GAACCCACAG 351 TTTCATTTCAATGGAACCAA AGGTGGAGTG GATCCAACAG CAAGTGGTGA 401 AAAAAAGAAC CAAGAGGGATTATGACCTCA GCCATGCCCA GTCAACCTAC 451 TTCAATGATC CCAAGTGGCC AAGTATGTGGTACATGCACT GCAGTGACAA 501 CACCCATCCT TGCCAGTCAG ACATGAATAT CGAAGGAGCCTGGAAGAGAG 551 GCTACACGGG GAAGAATATC GTGGTGACTA TCCTGGATGA CGGCATCGAG601 AGAACCCACC CAGATCTGAT GCAAAACTAC GATGCTCTGG CAAGTTGCGA 651TGTGAATGGG AATGACTTGG ACCCGATGCC TCGTTATGAT GCAAGCAATG 701 AGAACAAGCATGGGACCCGC TGTGCCGGAG AAGTGGCAGC CACTGCAAAC 751 AACTCTCACT GCACTGTCGGGATCGCTTTC AACGCCAAGA TTGGAGGTGT 801 GAGAATGCTG GATGGTGATG TCACTGACATGGTGGAGGCA AAGTCTGTCA 851 GCTACAACCC ACAGCATGTG CACATCTACA GTGCCAGCTGGGGACCAGAT 901 GACGATGGCA AGACTGTGGA TGGGCCAGCT CCCCTCACCC GGCAAGCCTT951 TGAGAATGGC GTGAGAATGG GGCGGAGAGG CCTTGGATCT GTGTTTGTGT 1001GGGCATCTGG CAATGGTGGA CGGAGCAAGG ATCACTGTTC TTGTGATGGC 1051 TATACCAACAGCATCTATAC CATCTCCATC AGCAGTACGG CCGAAAGTGG 1101 AAAGAAACCT TGGTACTTGGAAGAGTGTTC ATCTACACTG GCTACAACCT 1151 ACAGCAGTGG AGAATCCTAT GATAAGAAAATAATCACTAC TGATCTAAGG 1201 CAGCGATGCA CAGACAATCA CACTGGAACG TCAGCCTCAGCCCCCATGGC 1251 TGCTGGAATC ATTGCCCTGG CCCTAGAAGC CAATCCGTTT CTGACCTGGA1301 GAGACGTGCA GCATGTTATT GTCAGGACTT CCCGTGCGGG ACATTTGAAC 1351GCTAATGACT GGAAAACCAA TGCTGCTGGT TTTAAGGTGA GCCATCTCTA 1401 TGGATTTGGACTGATGGATG CCGAAGCCAT GGTGATGGAA GCAGAGAAGT 1451 GGACAACTGT TCCTCAGCAGCACGTGTGTG TGGAAAGCAC AGACCGACAA 1501 ATCAAGACCA TTCGACCAAA CAGTGCAGTGCGCTCCATCT ACAAAGCCTC 1551 AGGCTGCTCG GATAATCCCA ACCATCACGT CAATTACCTGGAGCATGTAG 1601 TTGTGCGTAT TACCATCACA CACCCACGGA GGGGAGACCT GGCCATCTAT1651 CTGACATCAC CCTCAGGAAC CAGATCCCAG CTCTTGGCCA ACAGGCTCTT 1701TGATCATTCC ATGGAAGGGT TTAAGAACTG GGAGTTCATG ACTATTCATT 1751 GCTGGGGAGAACGGGCTGCT GGGGACTGGG TCCTCGAAGT TTATGATACG 1801 CCATCTCAGC TGAGGAACTTCAAGACTCCA GGTAAATTGA AAGAATGGTC 1851 CTTAGTCCTC TATGGCACGT CCGTACAGCCATACTCCCCA ACCAACGAGT 1901 TTCCCAAAGT GGAACGCTTC CGCTACAGCC GAGTGGAAGACCCCACAGAT 1951 GACTACGGTG CTGAAGATTA TGCAGGTCCC TGTGACCCTG AATGCAGTGA2001 GGTTGGATGT GACGGGCCAG GACCAGATCA CTGCAGTGAC TGCTTACACT 2051ACTACTACAA GCTGAAAAAT AACACCAGAA TCTGTGTCTC CAGCTGCCCT 2101 CCTGGCCACTACCATGCTGA CAAGAAGAGG TGCCGGAAGT GTGCCCCAAA 2151 CTGCGAGTCC TGCTTTGGCAGCCATGGTGA TCAGTGCCTC TCCTGTAAAT 2201 ATGGCTACTT CCTGAATGAA GAAACTAGCAGCTGTGTTAC TCAGTGCCCT 2251 GATGGATCAT ACGAGGATAT CAAGAAAAAT GTCTGTGGGAAATGCAGTGA 2301 GAACTGCAAG GCATGCATTG GATTTCACAA CTGCACAGAG TGCAAGGGCG2351 GGTTAAGTCT TCAGGGATCC CGCTGTTCGG TCACCTGCGA GGATGGACAG 2401TTCTTCAATG GTCACGACTG CCAGCCCTGC CATCGCTTCT GTGCTACTTG 2451 CTCTGGGGCCGGAGCAGATG GATGTATTAA CTGCACCGAG GGGTATGTCA 2501 TGGAAGAGGG AAGGTGTGTACAAAGTTGTA GTGTGAGCTA CTACTTGGAC 2551 CACTCTTCAG AGGGTGGCTA CAAATCCTGCAAGAGATGTG ATAACAGCTG 2601 TTTGACATGC AATGGGCCAG GATTCAAGAA CTGTTCCAGCTGCCCCAGTG 2651 GATATCTTTT AGACTTAGGA ACGTGTCAGA TGGGAGCCAT CTGCAAGGAT2701 GCTACGGAAG AGTCCTGGGC AGAAGGAGGC TTCT

but is not able to hybridize under at least moderately stringentconditions to the sequences: 1 TGGAAGGCAA GGACTGGAAT GAAGCCGTGCCCACTGAAAA GCCATCTTTG SEQ ID NO: 5 51 GTGAGGAGTC TGCTGCAGGA TCGACGAAAGTGGAAAGTTC AAATCAAAAG 101 AGATGCAACG AGCCAGAATC AACCTTGTCA CTCTTCTTGTAAAACCTGCA 151 ATGGATCTCT CTGCGCTTCA TGTCCCACAG GTATGTACCT GTGGCTGCAG201 GCCTGTGTTC CTTCCTGTCC CCAAGGCACT TGGCCATCAG TCACCAGTGG 251CAGCTGTGAA AAGTGTTCCG AGGACTGTGT CTCCTGCTCC GGTGCCGACC 301 TTTGCCAACAGTGCCTGAGC CAGCCGGACA ACACTCTGCT TCTCCATGAG 351 GGCAGGTGCT ACCACAGTTGCCCAGAG; or 1 AGGGAGGCTG AGTTTTACGA GCACACCAAG ACTGCTCTGC TAGTGACCTC SEQID NO: 6 51 TGGCGCCATG CTGTTGCTGC TGCTGGGGGC TGCTGCGGTC GTGTGGCGGA 101AGTCTCGAAG CAGACCTGTG GCAAAGGGGC GGTACGAAAA GCTGGCAGAA 151 CCTACCGTGTCATACTCCTC CTACAGGAGC AGCTATCTTG ACGAGGACCA 201 GGTGATTGAG TACAGGGACCGGGACTACGA TGAGGACGAT GAGGACGACA 251 TCGTCTATAT GGGCCAAGAT GGCACTGTCTACCGGAAGTT CAAGTATGGG 301 CTGCTGGATG AGACGGAAGA TGATGAGTTG GAGTACGATGATGAGAGCTA 351 CTCTTACCAA TAAACAGAGC CCCCTCCCAT CTCAAACCCA CCACCCAC.

At the protein level, this 5.5 kb transcript encodes a PC 5/6 proteinwhich has a different C-terminal sequence for those of the knownisoforms of PC6A or PC6B.

The nucleic acid may be a cDNA, a genomic DNA, or an RNA and may be inthe sense or the anti-sense orientation. Preferably the nucleic acidmolecule is a cDNA.

Preferably the nucleic acid molecule comprises a sequence selected fromthe group consisting of:

-   -   (a) a cDNA molecule having the sequence set out in SEQ. ID. NO:        1 or SEQ ID NO: 2, or its human homologue (SEQ. ID. NO: 3);    -   (b) a nucleic acid molecule which is able to hybridise under at        least moderately stringent conditions to (a); and    -   (c) a nucleic acid molecule which has at least 75% sequence        identity to (a).

More preferably in (b) the nucleic acid molecule is able to hybridiseunder stringent conditions to the molecule of (a). More preferably in(c) the nucleic acid molecule has at least 80%, even more preferably atleast 90% sequence identity to the molecule of (a).

In a twelfth aspect, the invention provides a protein having proproteinconvertase 5/6 activity, which is encoded by a nucleic acid according tothe invention.

It will be clearly understood that for the purposes of the inventiondifferent isoforms of PC 5/6 may be suitable. Preferably in all aspectsof the invention, an isoform of PC 5/6 which is specifically expressedin implantation sites of the uterus during embryo implantation is used.

Defining appropriate hybridisation conditions is within the skill of theart. See e.g. Maniatis et al. DNA cloning volumes 1 and 11. Nucleic AcidHybridisation. Briefly, “moderately stringent conditions” forhybridisation or annealing of nucleic acid molecules are those which

-   -   (1) employ moderate ionic strength and moderate temperature for        washing, for example 0.3 M NaCl/0.03 M sodium citrate/0.1%        sodium dodecyl sulfate (SDS) at 42(C, or    -   (2) employ during hybridisation a denaturing agent such as        formamide, for example 40% (vol/vol) formamide with 0.1% bovine        serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium        phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium        citrate at 40(C.

Another example is use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 Msodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50(g/mL), 0.1% SDS, and 10% dextran sulfate at 42(C, with washes at 42(Cin 0.2×SSC and 0.1% SDS.

In a thirteenth aspect, the invention provides a composition comprisinga protein according to the invention, together with a pharmaceuticallyacceptable carrier.

Thus the invention provides a method of identifying agonists andantagonists of PC 5/6. In view of the crucial role in implantationindicated by the results reported herein, it is contemplated thatantagonists of PC 5/6 will be useful as contraceptives, and thatagonists of PC 5/6 will be useful as agents for promoting fertility orfor supporting at least the early phases of pregnancy. It is furthercontemplated that suitable antagonists of PC 5/6 include, but are notlimited to, antibodies and anti-sense nucleic acids. For example, aninhibitory antisense 17mer oligonucleotide is disclosed in InternationalPatent Application No. PCT/CA97/00535 (WO98/04686) by Day et al.

As used herein, the term “receptive” refers to a state of the uterinelining which will allow an embryo to implant, whereas a “non-receptive”state refers to a state in which attachment and implantation areimpaired. This can include a state known as “pre-receptive”.

The term “PC 5/6 antagonist” or “antagonist” refers to a substance whichopposes or interferes with a functional activity of PC 5/6.

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the results of RNA differential display analysis (DDPCR)of pregnant mouse uterus. The expression pattern of band 9 to beidentified as PC 5/6 on the DDPCR gel is indicated by the arrow, showingmuch stronger intensities in implantation sites (Imp) compared tointerimplantation sites (Inter) in three different mice: lane 1, animal1; lane 2, animal 2 and lane 3, animal 3.

FIG. 1B shows the results of Northern blot analysis of mRNA detected bythe cDNA extracted from band 0.9 of the DDPCR gel and used as a probe.Total RNA (15 μg) was isolated from implantation (Imp) andinterimplantation sites (Inter) of day 4.5 pregnant mice. The top panelshows multiple bands, mainly at −2.2 kb and ˜3.5 kb positions; the lowerpanel shows the signal detected by the GAPDH probe on the same membraneas in the top panel.

FIG. 2 shows the results of Northern blot analysis of mRNA detected bythe MUSPC6-fragB cDNA in the mouse uterus during early pregnancy and inthe intestine. Total RNA (15 μg) was isolated from whole uterus ofnon-pregnant mice at estrus (NP) and 3.5 day pregnant (d3.5) mice, andfrom implantation sites (Imp) and interimplantation sites (Inter) on day(d) 4.5, 5.5, 6.5, 8.5 and 10.5 of pregnancy. On day 8.5 and 10.5, threetypes of tissue were sampled:

-   -   (1) the entire implantation unit containing the uterine        implantation site, embryo and placenta [Imp (+)],    -   (2) uterine implantation site tissue without embryo and placenta        [Imp(−)], and    -   (3) embryo and placenta sampled together (Emb+P1).

The top panel shows the multiple transcripts (2.2 kb, 3.5 kb, 5.5 kb,6.5 kb and 10 kb) detected by this cDNA, and the lower panel shows thesignal detected by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)probe on the same membrane.

FIG. 3 shows the results of Northern blot analysis of total RNA (15 μg)isolated from whole uterus of non-pregnant mice at metestrus (met),diestrus (die), proestrus (pro) and estrus (est). One typical cycle isshown. The top panel shows the signals detected by the MUSPC6-fragBcDNA; the lower panel shows that detected by the GAPDH probe on the samemembrane.

FIG. 4 shows the results of Northern blot analysis of total RNA (15 μg)isolated from whole uterus of non-pregnant mice at estrus (NP), fromimplantation sites (Imp) and interimplantation sites (Inter) of day (d)4.5 pregnant mice, and from whole uterus of 4.5 pseudopregnant (Pseudopreg) mice. Day 0 is taken as the day of finding the vaginal plugresulting from copulation. The top panel shows signals detected by theMUSPC6-fragB cDNA; the lower panel shows that detected by the GAPDHprobe on the same membrane.

FIG. 5 shows the results of Northern blot analysis of the tissuespecificity of the MUSPC6 mRNA. Total RNA (15 μg) was isolated frommouse muscle, whole brain, kidney, spleen, heart, testis, ovary,implantation site on day 5.5 of pregnancy (d5.5-Imp), intestine, lung,liver and placenta. The top panel shows the signals detected byMUSPC6-fragB cDNA, and the lower panel shows the signal detected byribosomal 18s RNA probe on the same membrane.

FIG. 6 shows the results of Northern blot analysis performed to comparethe mRNA transcript profiles between the uterus and the intestine. TotalRNA (15 μg) was isolated from whole uterus of non-pregnant mice atestrus (NP), and from implantation sites (Imp) and interimplantationsites (Inter) on day (d) 4.5 and 5.5 of pregnancy. Total RNA (25 μg) wasalso isolated from the mouse intestine. The top panels show the mRNApatterns detected by the cDNA fragments representing the differentdomains of the MUSPC6 proteins, and the lower panel shows the signaldetected by a GAPDH probe on the same membrane. (A) The multipletranscripts detected by MUSPC6-fragA/DA/DB/G/H cDNA; (B) The 3.5 kbtranscript detected by MUSPC6-fragI; (C) The 6.5 kb transcript detectedby MUSPC6-fragK/M.

FIG. 7 shows the results of Northern blot analysis of the tissuespecificity of the MUSPC6B mRNA. Total RNA (15 μg) was isolated frommouse muscle, whole brain, kidney, spleen, heart, ovary, testis, liver,lung, intestine, and placenta, and from implantation sites (Imp) andinterimplantation (Inter) sites on day 4.5 of pregnancy. The top panelshows the 6.5 kb transcript detected by MUSPC6-fragK/M cDNA, and thelower panel shows the signal detected by ribosomal 18s RNA probe on thesame membrane.

FIG. 8 shows the results of Southern blot analysis of MUSPC6 DNA in themouse. Genomic DNA was isolated from non-pregnant mouse uterus, and 10μg was digested with the following four restriction enzymes: BamH1,EcoR1, HindIII and Bg1II probe with radio-labeled MUSPC6-fragH cDNA.

FIG. 9 shows the results of in situ hybridisation of MUSPC6 mRNA in asection of mouse uterus at an implantation site on day 4.5 of pregnancy.Artificially decidualized uterus was also tested.

FIG. 10 shows an MTE array (Clontech) demonstrating expression of PC6mRNA in a range of human tissues, including the uterus, heart,gastrointestinal tract, kidney, thymus, lung, placenta, testis andovary. The probe was a human PC6 cDNA.

FIG. 11 shows the results of Northern blot analysis of a range of humantissues using a human PC6 cDNA probe. Total RNA (10 g) was applied toeach lane. The probe was a human PC6 cDNA.

FIG. 12 shows results of RT-PCR for PC6 on human endometrial samplestaken across the normal menstrual cycle.

FIG. 13 shows results of RT-PCR of human PC6 in early pregnant humantissue (first trimester decidua and placenta), heart, perimenopausalovary, post-menopausal ovary, and term placenta.

FIG. 14 shows results of real-time quantitative RT-PCR analysis of humanPC6 mRNA from human endometrial cancer tissues (stages 1-3).

DETAILED DESCRIPTION OF THE INVENTION

PC 5/6 may be used as an immunogen to generate anti-PC 5/6 antibodies.Such antibodies, which specifically bind to PC 5/6, are useful in assaysfor PC 5/6, such as radioimmunoassay, enzyme-linked immunoassay, orcompetitive-type receptor binding assay or radioreceptor assay, as wellas in affinity purification techniques. The anti-PC 5/6 antibody shouldpreferably bind PC 5/6 with an affinity of at least about 10⁶ L/mole,and more preferably at least about 10⁷ L/mole.

Polyclonal antibodies directed toward PC 5/6 are generally raised inanimals by multiple subcutaneous or intraperitoneal injections of PC 5/6and an adjuvant. If necessary, immunogenicity may be increased byconjugating PC 5/6 or a peptide fragment thereof to a carrier proteinwhich is immunogenic in the species to be immunized, such as keyholelimpet hemocyanin, serum albumin, bovine thyroglobulin, soybean trypsininhibitor, or pertussis toxin, using a bifunctional or derivatizingagent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (conjugation throughlysine residues), glutaraldehyde, succinic anhydride, SOCl2, orR¹N═C═NR, where R and R¹ are different alkyl groups.

It will be appreciated that a fragment or derivative of PC 5/6 whichretains the ability to elicit anti-PC 5/6 antibody may alternatively beused as the immunogen.

Animals such as rabbits, sheep or mice are typically immunized with suchPC 5/6-carrier protein conjugates combining 1 mg or 100 μg of conjugate(for rabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant or another appropriate adjuvant known to those skilled in theart (eg. Montanide: Marcol), and injecting the solution intradermally atmultiple sites. One month later the animals are boosted with {fraction(1/5)}th to {fraction (1/10)}th the original amount of conjugate inFreund's complete adjuvant (or other appropriate adjuvant) bysubcutaneous injection at multiple sites. 7 to 14 days later animals arebled and the serum is assayed for anti-PC 5/6 antibody titre. Animalsare boosted until the antibody titre plateaus. Preferably, the animal isboosted by injection with a conjugate of the same PC 5/6 with adifferent carrier protein and/or through a different cross-linkingagent. Conjugates of PC 5/6 and a suitable carrier protein also can bemade in recombinant cell culture as fusion proteins. Also, aggregatingagents such as alum are used to enhance the immune response.

Monoclonal antibodies directed toward PC 5/6 are produced using anymethod which provides for the production of antibody molecules bycontinuous cell lines in culture. The modifier “monoclonal” indicatesthe character of the antibody as being-obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Examplesof suitable methods for preparing monoclonal antibodies include theoriginal hybridoma method of Kohler et al 1975, and the human B-cellhybridoma method, Kozbor, 1984; Brodeur et al., 1987.

The monoclonal antibodies of the invention specifically include“chimeric” antibodies (immunoglobulins) in which a portion of the heavyand/or light chain is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (Cabilly et al., U.S. Pat.No. 4,816,567; Morrison et al 1984).

In a preferred embodiment, the chimeric anti-PC 5/6 antibody is a“humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.These non-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.

Humanization can be performed following methods known in the art (Joneset al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988), bysubstituting rodent complementarity-determining regions (CDRs) for thecorresponding regions of a human antibody. Alternatively, it is nowpossible to produce transgenic animals (e.g., mice) which are capable,upon immunization, of producing a full repertoire of human antibodies inthe absence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy-chainjoining region (JH) gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. See, for example, Jakobovits et al., 1993; Jakobovits et al.,1993; Bruggermann et al., 1993. Human antibodies can also be produced inphage-display libraries (Hoogenboom et al., 1991; Marks et al., 1991).

For diagnostic applications, anti-PC 5/6 antibodies typically will belabeled with a detectable moiety. The detectable moiety can be any onewhich is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³³P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; radioactive isotopic labels, such as ¹²⁵I, ³²P,³³P, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby David et al., 1974; Pain et al., 1981; Bayer et al., 1990.

The anti-PC 5/6 antibodies may be employed in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays (Zola, 1987).

Competitive binding assays rely on the ability of a labeled standard(e.g., PC 5/6 or an immunologically reactive portion thereof) to competewith the test sample analyte (PC 5/6) for binding with a limited amountof antibody. The amount of PC 5/6 in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex (David et al., U.S. Pat. No. 4,376,110). The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody whichis labeled with a detectable moiety (indirect sandwich assay). Forexample, one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

Neutralizing anti-PC 5/6 antibodies are useful as antagonists of PC 5/6.The term “neutralizing anti-PC 5/6 antibody” as used herein refers to anantibody which is capable of specifically binding to PC 5/6, and whichis capable of substantially inhibiting or eliminating the functionalactivity of PC 5/6 in vivo or in vitro. Typically a neutralizingantibody will inhibit the functional activity of PC 5/6 by at leastabout 50%, and preferably greater than 80%, as determined, for example,by an in vitro receptor binding assay.

PC 5/6 is believed to be useful in promoting the implantation of thefertilized egg, development of the placenta and the embryo, andmaintenance of pregnancy. Accordingly, PC 5/6 may be utilized in methodsfor the diagnosis and/or treatment of a variety of fertility-relatedconditions, including infertility due to luteal phase defect,infertility due to failure of implantation, pre-eclampsia, earlyabortion, intrauterine growth restriction (IUGR), abnormal uterinebleeding, endometriosis, and early parturition, and it may provide apotential target for contraception. It is also contemplated that it maybe implicated in other conditions, such as cancers, and in particularcancers of the reproductive system, such as endometrial cancer.

PC 5/6 may be formulated with other ingredients such as carriers and/oradjuvants, e.g. albumin, nonionic surfactants and other emulsifiers.There are no limitations on the nature of such other ingredients, exceptthat they must be pharmaceutically acceptable, efficacious for theirintended administration, and cannot degrade the activity of the activeingredients of the compositions. Suitable adjuvants include collagen orhyaluronic acid preparations, fibronectin, factor XIII, or otherproteins or substances designed to stabilize or otherwise enhance theactive therapeutic ingredient(s).

Animals or humans may be treated in accordance with this invention. Itis possible but not preferred to treat an animal of one species with PC5/6 of another species.

A number of inhibitors of proprotein convertases have been described.These include alpha₁-antitrypsin Portland (Jean et al, 1998) andN-terminal prosegments of the PCs which inhibit the parent enzyme(PC1/3, Boudreault et al 1998; Furin and PC7, Zhong et al, 1999).Although these are either non-specific or not shown for PC 5/6, similarinhibitors could be prepared which would be useful for the applicationsdescribed herein.

PC 5/6 and PC 5/6 antagonists to be used for in vivo administration mustbe sterile. This is readily accomplished by filtration of a solution ofPC 5/6 or anti-PC 5/6 antibody through sterile filtration membranes.Thereafter, the filtered solution may be placed into a container havinga sterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle. Thefiltered solution also may be lyophilized to produce sterile PC 5/6 oranti-PC 5/6 antibody in a powder form.

Methods for administering PC 5/6 and PC 5/6 antagonists, such assynthetic inhibitors, in vivo include injection or infusion byintravenous, intraperitoneal, intracerebral, intrathecal, intramuscular,intraocular, intraarterial, intravaginal, intrauterine, intracervical,or intralesional routes, and by means of sustained-release formulations,including suppositories.

Sustained-release formulations generally consist of PC 5/6 or PC 5/6antagonists and a matrix from which the PC 5/6 or PC 5/6 antagonists arereleased over some period of time. Suitable matrices includesemipermeable polymer matrices in the form of shaped articles, forexample, membranes, fibers, or microcapsules. Sustained release matricesmay comprise polyesters, hydrogels, polylactides, U.S. Pat. No.3,773,919, copolymers of L-glutamic acid and gamma ethyl-L-glutamate,Sidman et al., 1983, poly(2-hydroxyethyl-methacrylate), or ethylenevinyl acetate, Langer et al., 1981 & 1982. In one embodiment of theinvention, the therapeutic formulation comprises PC 5/6 or a PC 5/6antagonist entrapped within or complexed with liposomes. For example, PC5/6 covalently joined to a glycophosphatidyl-inositol moiety may be usedto form a liposome comprising PC 5/6.

An effective amount of PC 5/6 or PC 5/6 antagonist, e.g., anti-PC 5/6antibody, to be employed therapeutically will depend upon thetherapeutic objectives, the route of administration, and the conditionof the patient. Accordingly, it will be necessary for the therapist totitrate the dosage and modify the route of administration as required toobtain the optimal therapeutic effect. A typical daily dosage mightrange from about 1 μg/kg to up to 100 mg/kg or more, depending on thefactors mentioned above. Much lower doses may be possible if the agentis administered locally. Where possible, it is desirable to determineappropriate dosage ranges first in vitro, for example, using assays forserine protease activity or specific PC 5/6 activity which are known inthe art, and then in suitable animal models, from which dosage rangesfor human patients may be extrapolated.

For example, the dose of a protein PC 5/6 antagonist, particularly anantibody, can be about 0.1 mg to about 500 mg, typically about 1.0 mg toabout 300 mg, more typically about 25 mg to about 100 mg. Theadministration frequency can be appropriately selected depending uponthe condition to be treated and the dosage form for the desiredtherapeutic effects.

The mammal may be a human, or may be a domestic or companion animal.While it is particularly contemplated that the compounds of theinvention are suitable for use in medical treatment of humans, they arealso applicable to veterinary treatment, including treatment ofcompanion animals such as dogs and cats, and domestic animals such ashorses, cattle and sheep, or zoo animals or feral mammals such asnon-human primates, felids, canids, bovids, and ungulates.

Methods and pharmaceutical carriers for preparation of pharmaceuticalcompositions are well known in the art, as set out in textbooks such asRemington's Pharmaceutical selected with the planned manner ofadministration in mind.

The compounds of the invention may be administered orally, topically, orparenterally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.The term parenteral as used herein includes subcutaneous, intravenous,intramuscular, intrathecal, intracranial, injection or infusiontechniques.

The invention also provides suitable topical, oral, aerosol, andparenteral pharmaceutical formulations for use in the novel methods oftreatment of the present invention. The compounds of the invention maybe administered orally as tablets, aqueous or oily suspensions,lozenges, troches, powders, granules, emulsions, capsules, syrups orelixirs. The composition for oral use may contain one or more agentsselected from the group of sweetening agents, flavouring agents,colouring agents and preserving agents in order to producepharmaceutically elegant and palatable preparations. The tablets containthe active ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.

These excipients may be, for example, inert diluents, such as calciumcarbonate, lactose, calcium phosphate or sodium phosphate; granulatingand disintegrating agents, such as corn starch or alginic acid; bindingagents, such as starch, gelatin or acacia; or lubricating agents, suchas magnesium stearate, stearic acid or talc. The tablets may beuncoated, or may be coated by known techniques to delay disintegrationand absorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time-delaymaterial such as glyceryl monostearate or glyceryl distearate may beemployed. Coating may also be performed using techniques described inthe U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotictherapeutic tablets for controlled release.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers such as those based on Ringer's dextrose, and the like.Preservatives and other additives may also be present, such asanti-microbials, anti-oxidants, chelating agents, growth factors andinert gases and the like.

Generally, the terms “treating”, “treatment” and the like are usedherein to mean affecting a subject, tissue or cell to obtain a desiredpharmacological and/or physiological effect. The effect may beprophylactic in terms of completely or partially preventing a disease orsign or symptom thereof, and/or may be therapeutic in terms of a partialor complete cure of a disease. “Treating” as used herein covers anytreatment of, or prevention of disease in a vertebrate, a mammal,particularly a human, and includes: preventing the disease fromoccurring in a subject who may be predisposed to the disease, but hasnot yet been diagnosed as having it; inhibiting the disease, ie.,arresting its development; or relieving or ameliorating the effects ofthe disease, ie., causing regression of the effects of the disease.

The invention includes various pharmaceutical compositions useful forameliorating disease. The pharmaceutical compositions according to oneembodiment of the invention are prepared by bringing a compound offormula I, analogue, derivatives or salts thereof and one or morepharmaceutically-active agents or combinations of compound of formula Iand one or more pharmaceutically-active agents into a form suitable foradministration to a subject using carriers, excipients and additives orauxiliaries.

Frequently used carriers or auxiliaries include magnesium carbonate,titanium dioxide, lactose, mannitol and other sugars, talc, milkprotein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 20th ed. Williams & Wilkins (2000) and The British NationalFormulary 43rd ed. (British Medical Association and Royal PharmaceuticalSociety of Great Britain, 2002; http://bnf.rhn.net), the contents ofwhich are hereby incorporated by reference. The pH and exactconcentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's, The Pharmacological Basis for Therapeutics (7thed., 1985).

The pharmaceutical compositions are preferably prepared and administeredin dosage units. Solid dosage units include tablets, capsules andsuppositories. For treatment of a subject, depending on activity of thecompound, manner of administration, nature and severity of the disorder,age and body weight of the subject, different daily doses can be used.Under certain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

The pharmaceutical compositions according to the invention may beadministered locally or systemically in a therapeutically effectivedose. Amounts effective for this use will, of course, depend on theseverity of the condition and the weight and general state of thesubject. Typically, dosages used in vitro may provide useful guidance inthe amounts useful for in situ administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of the cytotoxic side effects. Variousconsiderations are described, eg., in Langer, Science, 249: 1527,(1990). Formulations for oral use may be in the form of hard gelatincapsules, in which the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin.They may also be in the form of soft gelatin capsules, in which theactive ingredient is mixed with water or an oil medium, such as peanutoil, liquid paraffin or olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspension. Suchexcipients may be suspending agents such as sodium carboxymethylcellulose, methyl cellulose, hydroxypropylmethylcellulose, sodiumalginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents, which may be (a) a naturally occurringphosphatide such as lecithin; (b) a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate; (c) acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol; (d) a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand hexitol such as polyoxyethylene sorbitol monooleate, or (e) acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to known methods using suitable dispersing orwetting agents and suspending agents such as those mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents which may be employed are water, Ringer'ssolution, and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed, includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid may be used in the preparation of injectables.

The compounds of the invention may additionally be combined with othercompounds to provide an operative combination. It is intended to includeany chemically compatible combination of pharmaceutically-active agents,as long as the combination does not eliminate the activity of theprotein of this invention.

The invention will now be described in detail by way of reference onlyto the following non-limiting examples and drawings.

Materials and Methods

Animals and Tissue Preparation

Swiss outbred mice were housed and handled according to the MonashUniversity animal ethics guidelines on the care and use of laboratoryanimals. All experimentation was approved by the Institutional AnimalEthics Committee at the Monash Medical Centre. Adult female mice (6-8weeks old) were mated with fertile males of the same strain to producenormal pregnant animals, or mated with vasectomized males to producepseudopregnant mice. The morning of finding a vaginal plug wasdesignated as day 0 of pregnancy. Uterine tissues were collected fromnon-pregnant mice, or from pregnant mice on days 3-11. A selection ofother mouse organs was also collected from non-pregnant mice. Tissueswere snap-frozen in liquid nitrogen for Northern analysis, or fixed in4% buffered formalin (pH 7.6) for in situ hybridisation.

For non-pregnant and day 3.5 pregnant mice, the entire uterus wascollected. For day 4.5 pregnant mice, implantation sites were visualisedby intravenous injections of a Chicago Blue dye solution (1% in saline,0.1 ml/mouse) into the tail vein 5 min before killing the animals;implantation sites were separated from interimplantation sites, and bothsites were retained. For pregnant mice on day 5.5 onwards, implantationand interimplantation sites were visualized without dye injection.

For non-pregnant mice, the uterus was also collected from differentstages of the estrous cycle: metestrus, diestrus, proestrus and estrus.The stages of the cycle were determined by analysis of vaginal smears(Rugh, 1994). For ovarian hormone treatments, the animals were firstovariectomized under anaesthesia with avertin, without regard to thestage of the estrous cycle (Rugh, 1994).

EXAMPLE 1 RNA Differential Display (DDPCR) and Isolation of cDNAs

DDPCR was performed as previously described (Nie et al, 2000b), and asdescribed originally by Liang and Pardee (1992 & 1993).

Total RNA was extracted from pools of implantation or interimplantationsite tissues of mice by acid guanidinium thiocyanate-phenol-chloloroformextraction (Chomczynski and Sacchi, 1987). The RNA was then treated withRNase-free DNase in the presence of placental RNase inhibitor to removeany contaminating DNA. The amount of RNA in the final preparation wasdetermined spectrophotometrically, and the RNA quality was evaluated bythe ratio of OD260 over OD280 (>1.8). DNA-free RNA (1(g) from theimplantation and interimplantation sites of three animals was used asthe template for the first-strand cDNA synthesis in a 20 μl reactionmixture in the presence of 20 μM dNTPs, 50 μM oligo-dT anchored primers(one of T12MG, T12MC, T12MA and T12MT), as shown in Table 1, 10 mM DTT,10 U RNasin ribonuclease inhibitor (Promega, Madison, Wis.), 25 U AMVreverse transcriptase (Boehringer Mannheim, Nunawading, Victoria,Australia), and the cDNA synthesis buffer (Boehringer Mannheim). Thesynthesised cDNA was then amplified by PCR in 20 μl with the followingcomponents: 2 μl of cDNA, 1×PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl₂, 50mM KCl, pH 8.3), 10 μM dNTPs, 10 picomoles of one random decamer (Table1, Operon 10-mer kit A; Operon, Alameda, Calif.), 50 picomoles of oneoligo-dT anchored primer (as used in cDNA synthesis), 2 (Ci of ³³P-dATP(Du Pont Ltd., North Sydney, Australia) and 1 U of Taq DNA polymerase(Boehringer Mannheim). The PCR was performed in a Hybaid OmniGene PCRsystem (Hybaid Ltd, Teddington, Middlesex, UK) with the followingconditions: initial denaturation at 94° C. for 5 min; then 40 cycles ofdenaturation at 94° C. for 30 sec, primer annealing at 39° C. for 2 min,and a final extension at 72° C. for 30 sec; and a final extension at 72°C. for 10 min. The subsequent PCR products (4 μl) were run on 6%high-resolution polyacrylamide/urea gel (Liang & Pardee, 1992) andvisualised by autoradiography. The majority of amplified fragments wereshared between the implantation and interimplantation sites. Bandsunique to implantation sites compared to interimplantation sites wereidentified as of interest. The 80 PCR primer combinations (20 random 10mers combined with 4 oligo-dT anchored primers) used in the initialDDPCR analysis are shown in Table 1.

The candidate bands obtained from the DDPCR analysis were excised fromthe dried sequencing gel, incubated with 100 μl H₂O and 2 drops of oilat 100° C. for 15 min, followed by room temperature incubation for 1 hr,and then the mixture was vigorously vortexed before centrifugation for 5min to remove debris and obtain the cDNA. The eluted cDNAs werereamplified by PCR using the conditions described above. Thedifferential display pattern was further confirmed by Northern blottinganalysis using as probes the reamplified PCR products, which weregenerated by random hexamer labelling. TABLE 1 The 80 (4 × 20) primercombinations used in DDPCR 3′ primers: Oligo-(dT) anchored primers,custom-made Primer Code Sequence SEQ ID NO. 1 T12MA TTTTTTTTTTTT(G,A,C)A7 2 T12MC TTTTTTTTTTTT(G,A,C)C 8 3 T12MG TTTTTTTTTTTT(G,A,C)G 9 4 T12MTTTTTTTTTTTTT(G,A,C)T 10

5′ Primers: 10 mers, from Operon SEQ Prim- ID er Code Sequence NO. 1OPA-01 CAGGCCCTTC 11 2 OPA-02 TGCCGAGCTG 12 3 OPA-03 AGTCAGCCAC 13 4OPA-04 AATCGGGCTG 14 5 OPA-05 AGGGGTCTTG 15 6 OPA-06 GGTCCCTGAC 16 7OPA-07 GAAACGGGTG 17 8 OPA-08 GTGACGTAGG 18 9 OPA-09 GGGTAACGCC 19 10OPA-10 GTGATCGCAG 20 11 OPA-11 CAATCGCCGT 21 12 OPA-12 TCGGCGATAG 22 13OPA-13 CAGCACCCAC 23 14 OPA-14 TCTGTGCTGG 24 15 OPA-15 TTCCGAACCC 25 16OPA-16 AGCCAGCGAA 26 17 OPA-17 GACCGCTTGT 27 18 OPA-18 AGGTGACCGT 28 19OPA-19 CAAACGTCGG 29 20 OPA-20 GTTGCGATCC 30

To avoid embryonic contamination, the embryos were removed under thelight microscope from the implantation sites. After the DDPCR analysis,the differential display pattern was further verified by the Northernblotting analysis and cDNAs from those confirmed bands were sub-clonedinto the pGEM-T vector (Promega, Madison, Wis., USA) and sequencedmanually.

For Northern blot analysis, no attempt was made to separate the embryosfrom the decidua before day 8 of pregnancy, but for 8- and 11-daypregnant mice, embryos were separated from the uterine tissue. Total RNAwas extracted from whole uteri or from pools of implantation orinter-implantation sites by the acid guanidiniumthiocyanate-phenol-chloroform extraction (GTC) method (Chomczynski &Sacchi, 1987). RNA (10-15 (g) was denatured at 50° C. for 60 min in 50%dimethylsulfoxide (DMSO) and 1M glyoxal, and the denatured RNA wasfractionated by electrophoresis through a 1.2% agarose gel in 10 mMsodium phosphate buffer (pH 7.0) and transferred to positively chargednylon membranes (Hybond-N+, Amersham) by overnight capillary blotting in5×SSPE (1×SSPE=150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4). Membraneswere baked at 80° C. for 2 h followed by 3 min UV cross linking.

Transcript size was estimated by comparison with RNA size standards(Gibco-BRL, Gaithersburg, Md. USA). A simplified filter paper sandwichblotting method (Jones & Jones, 1992; Nie et al, 2000b) was used for thehybridisation process at 42° C. overnight, without a prehybridisationstep. The hybridisation buffer contained 50% formamide, 6×SSPE (900 mMsodium chloride, 60 mM sodium phosphate, pH 7.4, 6 mM EDTA), 5×Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovineserum albumin), 0.5% SDS, 0.1 mg/ml sheared herring sperm DNA, and³²P-dCTP-labelled probes. The radio-labeled cDNA probes were generatedby random primer labeling of 25 ng cDNA with [³²P]deoxy-CTP (50(Ci/reaction).

Unincorporated nucleotides were removed with a MicroSpin S-200 HR column(AMRAD Pharmacia Biotech, Melbourne, Australia). Followinghybridisation, the blots were rinsed twice with 5×SSPE at 37° C., thentwice for 15 min each at 37° C. with 2×SSC/0.1% SDS (w/v) (1×SSC=150 mMNaCl, 15 mM Na₃ citrate, pH 7.4). In some cases, additional washes werealso performed with 0.5 or 1×SSC/0.1% SDS for 15 min at 60° C. Todetermine lane to lane loading variation, each blot was also probed witha mouse cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)or 18S ribosomal RNA. Between hybridisations, blots were stripped byincubation at 80° C. for 3 h in 1 mM EDTA/0.1% SDS followed by rinsingin H₂O.

For reverse transcriptase-polymerase chain reaction (RT-PCR), 1 μgDNA-free total RNA was reverse-transcribed at 46° C. for 1-1.5 h in 20μl reaction mixture, using 100 ng random hexanucleotide primers and AMVreverse transcriptase (Boehringer-Mannheim, Nunawading, Vic., Australia)with the cDNA synthesis buffer. The PCR was performed in a total volumeof 40 μl with 1-1.5 μl of the RT reaction, 1×PCR buffer, 20 μM dNTPs, 10pmol forward and reverse primers and 2.5 units of Taq DNA polymerase(Boehringer-Mannheim), in 3 stages as follows:

-   -   (a) one cycle of an incubation for 5 min at 95° C., 1 min at 52°        C.-60° C., and 2 min at 72° C.;    -   (b) 32 cycles with a denaturation for 45 sec at 95° C.,        annealing at 52° C.-60° C. for 50 sec and extension at 72° C.        for 1 min; and    -   (c) incubation for 5 min at 72° C.

The PCR products were subjected to electrophoresis (100 v/0.5×TBEbuffer) on 1.5% agarose gel, and stained with ethidium bromide. Bands ofinterest were cut out from the agarose gels, purified with the Qiaquickgel extraction kit (Qiagen Pty Ltd., Clifton Hill, Vic., Australia),cloned into a pGEM-T easy vector (Promega) according to themanufacturer's instructions and sequenced on an automated sequencer(Applied Biosystems, ABI Prism™, 377 DNA Sequencer) using the ABI PrismBigDye terminator cycle sequencing ready reaction kit.

The primer sequences used to amplify the following fragments of mouseproprotein convertase PC6 (MUSPC6 GenBank Accession No.: D12619; 2994bp)) from mouse uterine RNA were:

(1) MUSPC6-fragA (171 bp), upper primer 5′ GTT AGT TGT GCG CGC TGC TTA3′ SEQ ID NO: 31 [nucleotide (nt) 4-24]

and lower primer 5′ GCG CGT CCG GCA TAC C 3′ SEQ ID NO: 32 (nt 159-174)

(2) MUSPC6-fragB (422 bp), upper primer 5′ CAC CCA TCC TTG CCA GTC AG 3′SEQ ID NO: 33 (nt 501-520)

and lower primer 5′ CAT CCA CAG TCT TGC CAT CGT 3′ SEQ ID NO: 34 (nt902-922).

(3) MUSPC6-fragD (656 bp), upper primer 5′ GAT CGG TGC ACT GAA GGA CTA3′ SEQ ID NO: 35 (nt 267-287)and lower primer

-   -   5′ CAT CCA CAG TCT TGC CAT CGT 3′ (nt 902-922);    -   this fragment was digested with HindIII, and two smaller        fragments (177 bp and 479 bp) were obtained; the 177 bp was then        designated as MUSPC6-fragDA and the 479 bp as MUSPC6-fragDB.

MUSPC6-fragG (653 bp), upper primer 5′ CTG GCA GCA TGT TAT TGT CA 3′ SEQID NO: 36 (1305-1324)

and lower primer 5′ CGT AGT CAT CTG TGG GGT CTT 3′ SEQ ID NO: 37(1937-1957).

(5) MUSPC6-fragH (672 bp), upper primer 5′ CCA CTA CCA TGC TGA CAA GA 3′SEQ ID NO: 38 (nt 2106-2125)

and lower primer 5′ AGA ACT TTT CGC TGA CAG AGA 3′ SEQ ID NO: 39 (nt2757-2777)

(6) MUSPC6-fragI (227 bp), upper primer 5′ GTA TGC TTG TGA AAA AGA ACA3′ SEQ ID NO: 40 (nt 2735-2755)

and lower primer 5′ CCC TGC ACA AAC TTG AGA TAG 3′ SEQ ID NO: 41 (nt2941-2961)

The following two fragments were cloned by RT-PCR from mouse intestineRNA on the basis of the MUSPC6B cDNA sequence (Accession: D17583, 5208bp):

(1) MUSPC6-fragK (377 bp), upper primer 5′ TGG AAG GCA AGG ACT GGA ATG3′ SEQ ID NO: 42 (nt 2522-2542)

and lower primer 5′ CTC TGG GCA ACT TGT GTA GCA 3′ SEQ ID NO: 43 (nt2876-2898)

(2) MUSPC6-fragM (398 bp), upper primer 5′ AGG GAG GCT GAG TTT TAC GAG3′ SEQ ID NO: 44 (nt 4285-4305)

and lower primer 5′ GTG GGT GGT GGG TTT GAG 3′ SEQ ID NO: 45 (nt4665-4682).

EXAMPLE 2 Analysis of MUSPC6 Gene by Southern Blotting

Total genomic DNA was isolated from non-pregnant uterus and from kidney,using the DNeasy Tissue Kit (Qiagen Pty Ltd). A total amount of 10 μgwas then digested separately with an excess of several restrictionendonucleases (BamH1, EcoR1, HindIII, and BglII) at 37° C. for 14 hours,and fractionated on 0.8% agarose gel. The DNA was then blotted on topositively charged nylon membranes (Hybond-N, Amersham) using thestandard Southern blotting procedure (Sambrook et al. 1989) and probedwith radio-labeled cDNA, as described for the Northern analysis.

EXAMPLE 3 Artificial Decidualization of Mouse Uterus and In SituHybridisation

To induce artificial decidualization in the mouse uterus, non-pregnantmice were ovariectomised and treated with estradiol and progesterone asdescribed by Finn and Pope (Finn and Pope. 1994), and uteri collected 48hours after the administration of oil stimulus.

Sense and anti-sense digoxygenin (DIG)-labelled RNA probes against PC5/6 (clone 9.5) were generated using the DIG RNA Labeling kit(Boehringer Mannheim), and the concentrations determined according tothe manufacturer's instructions. Five micron sections of formalin-fixedparaffin-embedded tissues were subjected to in situ hybridisation asdescribed by (Komminoth, 1992). All sections were counterstained withMayer's hematoxylin.

EXAMPLE 4 DDPCR Analysis and Confirmation of Clone 9.5 by NorthernBlotting

To identify genes which are potentially critical for the initial processof embryo implantation in the mouse, we compared the gene expressionpattern of implantation and interimplantation sites in the uterus on day4.5 of pregnancy, using the DDPCR technique. Seventeen bands, of whichthe intensities were different between the two sites, were detected onDDPCR gels (Nie et al., 2000 One of these bands, band 9, was fullyanalysed.

On the DDPCR gel, band 9 was much more intense in implantation sitescompared to interimplantation sites in all individual animals tested asshown in FIG. 1A. To verify that this band indeed represents gene(s)which are differentially expressed between the two sites, the cDNAproducts of band 9 were extracted out of the DDPCR gel, re-amplified andcloned into the pGEM-T vector; Northern blot analysis was performedusing the cloned inserts as probes. Among the clones analysed, cDNA ofclone 9.5 explicitly detected differential expression of mRNA betweenthe two sites on the Northern blot, with much higher mRNA level presentin implantation sites compared to interimplantation sites. This isillustrated in FIG. 1B. On this initial Northern blot, more than onetranscript were observed, with a 3.5 kb band being the prominenttranscript. This confirmed that clone 9.5 contained the cDNArepresenting the original expression pattern of band 9 on the DDPCR gel.

EXAMPLE 5 Sequence Analysis of Clone 9.5 and Comparison with the GenbankDatabase

Band 9 resulted from the DDPCR amplification of day 4.5 implantationsite mRNA with the following two primers: 5′ primer, GTGACGTAGG (OPA-8,Table 1) and 3′ primer, T12MA (Table 1). After the confirmation thatclone 9.5 contained the cDNA representing band 9, the nucleotidesequence of this clone was determined, and is shown in Table 2. Itcontained 158 nucleotides, and the ends of the sequence indeed containedthe unique and expected primer sequence of GTGACGTAGG at the 5′ end, andthe reverse complementary sequence of T12MA at the 3′ end (underlined inTable 2). This confirmed that the cDNA in clone 9.5 was indeed thedirect PCR product amplified from the specific primers applied duringDDPCR amplification. TABLE 2 The sequence of clone 9.5 (158 bp) derivedfrom band 9 of DDPCR analysis 1 GTGACGTAGG ACAGATCGGT GCACTGAAGGACTACTATCA CTTCTACCAT SEQ ID NO: 46 51 AGTAGGACCA TTAAAAGGTC TGTTCTCTCGAGCAGAGGAA CCCACAGTTT 101 CATTTCAATG GAACCAAAGG TGGAGTGGAT CCAACAGCAAGTGGTGAAAA 151 AAAAAAAA* The underlined parts represent the primers used during DDPCRamplification

When compared to the GenBank database, the sequence of clone 9.5 showed98% identity to mouse serine protease PC6 (MUSPC6)(Accession: D12619;2994 bp), 98% identity to mouse convertase PC5 (MUSPC5)(Accession:L14932; 2848 bp) and 98% identity to human protease PC6 isoform A(HPC6A)(Accession: U56387; 2844 bp). It appears that in fact MUSPC5 andMUSPC6 are actually the same gene under different names, and that theyare 90% homologous to HPC6A cDNA. The detailed comparison of clone 9.5(SEQ ID NOS: 47 and 48) with MUSPC6 cDNA (SEQ ID NOS: 49 and 50) isillustrated in Table 3, showing that clone 9.5 aligned to nucleotide(nt) 254-411 of MUSPC6 cDNA. In principle, and in mostexperimentally-tested cases, a DDPCR-derived fragment should representthe 3′ end sequence for a cDNA (Liang & Pardee, 1992; Nie et al. 2000);however, in contrast to this expectation, clone 9.5 aligns with thenucleotide sequence 254-411, which is near the 5′ end, but not to the 3′end of MUSPC6 cDNA (Table 3). This unusual outcome is thought to resultfrom the unique sequence of MUSPC6 cDNA (Table 3) and the intrinsicnature of PCR, in which the 5′ end of a primer sequence can bedegenerate. TABLE 3 The comparison of nucleotide sequence of clone 9.5(158 bp) with mouse PC6 cDNA                                 10        20        30 clone9.5                           GTGACGTAGGACAGATCGGTGCACTGAAGGACT                         ::::::::::::::::::::::::::::::::::: MUSPC6ATCGCATAGCCAGCAAGTACGGATTCATCAACGTAGGACAGATCGGTGCACTGAAGGACT 230       240       250       260      270       280    100       110       120       130       140      150 clone9.5ACAGTTTCATTTCAATGGAACCAAAGGTGGAGTGGATCCAACAGCAAGTGGTGAAAAAAA:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: MUSPC6ACAGTTTCATTTCAATGGAACCAAAGGTGGAGTGGATCCAACAGCAAGTGGTGAAAAAAA 350       360       370       380       390       400 CLONE9.5 AAAAA :: MUSPC6 GAACC   411

EXAMPLE 6 RT-PCR Cloning of Mouse PC6 (MUSPC6) and Determination ofMUSPC6 mRNA Expression in the Uterus During Early Pregnancy

The results shown in FIG. 1B and Table 3 indicated that clone 9.5represents part of MUSPC6 cDNA, and that the level of expression of thisgene is much higher in implantation sites compared to theinterimplantation sites on day 4.5 of pregnancy. To confirm the identityof clone 9.5 and verify that MUSPC6 mRNA is indeed differentiallyexpressed between the two sites, two additional cDNA fragments ofMUSPC6, designated MUSPC6-fragA and MUSPC6-fragB, were cloned using theRT-PCR approach, and used as cDNA probes on the Northern blots.MUSPC6-fragA was a 171 bp fragment covering nucleotides 4-174, andMUSPC6-fragB was a 422 bp fragment covering nucleotides 501-922 ofMUSPC6 cDNA (Accession: D12619; 2994 bp). These two fragments weredesigned to represent the up- and down-stream region of MUSPC6 cDNA, towhich clone 9.5 is aligned. Using MUSPC6-fragA and MUSPC6-fragB asprobes, a pattern of mRNA expression similar to that shown in FIG. 1B,where clone 9.5 was used as a probe, was observed between implantationsites and interimplantation sites (data not shown). This conclusivelyconfirmed the identity of clone 9.5 as representing MUSPC6, and showedthat MUSPC6 mRNA is up-regulated in implantation sites on day 4.5 ofpregnancy.

In a subsequent experiment, MUSPC6-fragB was used as the cDNA probe, andadditional Northern analyses were performed to determine the expressionpattern of this gene in the uterus in relation to the time ofimplantation and early pregnancy. Total RNA from the uterus ofnon-pregnant mice (estrus) and pregnant mice at the initial stage forimplantation (day 4.5 of pregnancy) through to fully establishedimplantation and placentation (day 10.5 of pregnancy) was analysed. Theresults are shown in FIG. 2. Very low expression was observed innon-pregnant mice, as well as in mice on day 3.5 of pregnancy. However,around day 4.5 up to day 6.5 of pregnancy a dramatic upregulation ofthis gene occurred in the implantation sites, but not in theinterimplantation sites (FIG. 2). Beyond day 6.5 of pregnancy, theexpression level then drastically decreased, and returned to the levelin non-pregnant uterus.

Thus this upregulation was very transient, and only occurred between day4.5 and 6.5 of pregnancy, when the embryos are actively implanting intothe uterus. The upregulation was very implantation site-specific, anddid not occur uniformly along the uterine horns, but only in theimplantation sites.

In addition, it was noticeable that more bands were observed in FIG. 2than in FIG. 1B. This was simply due to the different lengths of filmexposure for the two Northern blots. After the experiment reported inFIG. 2, the result of FIG. 1B was repeated, and similar numbers of bandswere detected after much longer film exposure. Thus it is clear that inimplantation sites during the active embryo implantation period, theuterus expresses multiple transcripts of MUSPC6, with a size range of2.2 kb, 3.5 kb, 6.5 kb and 10 kb; of these the 3.5 kb transcript is themost abundant. This observation agrees with the finding that multipletranscripts were detected for MUSPC6 in the brain and intestine(Nakagawa et al. 1993 a & b).

EXAMPLE 7 MUSPC6 mRNA Level During the Estrous Cycle

The influence of the estrous cycle on the expression of MUSPC6 in thenon-pregnant uterus was examined by determining the level of MUSPC6 mRNAby Northern analysis. The study utilised 16 individual mice at differentstages of the cycle (metestrus, diestrus, proestrus and estrus), groupedto represent 4 cycles, and results from one typical cycle are shown inFIG. 3. In all cases, the expression level was very low at estrus andproestrus and became marginally higher during metestrus and diestrus;however, the levels at all stages of the estrous cycle were much lowerthan that in the implantation sites during the implantation period.These results indicated that the upregulation observed in implantationsites is not solely a direct result of the influence of the ovarianhormones during pregnancy, but requires other factors, such as thepresence of an embryo.

EXAMPLE 8 Effects of the Embryo on Uterine Expression of MUSPC6

To determine whether the presence of an embryo in the uterus wasessential for the observed changes in MUSPC6 mRNA expression duringearly pregnancy, total RNA was isolated from mice on day 4.5 ofpseudopregnancy, and the mRNA expression pattern compared with that innormal day 4.5 pregnant animals by Northern analysis. The results areshown in FIG. 4. This experiment demonstrated that the mRNA level in thepseudopregnant mice was actually equivalent to that in interimplantationsites of pregnant animals, and was much lower than that at theimplantation sites. This implied that the observed increase in MUSPC6mRNA expression at the implantation sites during early pregnancyrequired the presence of embryos in the uterus.

EXAMPLE 9 Tissue Distribution of MUSPC6 mRNA

Multi-tissue Northern analysis was performed to investigate the tissuedistribution of MUSPC6 mRNA expression. The results, illustrated in FIG.5, show that MUSPC6 is not widely expressed. When an equal amount oftotal RNA was compared, the implantation sites on day 5.5 showed thehighest level of expression, and apart from the uterus, only intestine,ovary, testis and brain among the 12 tissues tested showed detectableexpression.

EXAMPLE 10 Evidence from a Uterine- and Pregnancy-Specific Form ofMUSPC6

MUSPC6 has been previously studied in the brain and intestine (Nakagawaet al. 1993 a & b); therefore intestine was chosen as the positivecontrol tissue for our studies. However, it was observed that theprofile of the mRNA transcripts detected by Northern blotting wasdifferent in the uterus from that in the intestine. Both tissues showmultiple transcripts ranging from 2.2 kb to 10 kb, but there is a subtledifference; the 6.5 kb transcript present in the intestine is replacedby a 5.5 kb one in the uterus, as shown in FIG. 2. This 6.5 kbintestinal transcript has been previously investigated in detail, andshown to encode a MUSPC6 isoform, designated as mouse PC6 B (Accession:D17583, 5208 bp), which has an extremely large cysteine-rich motif(Nakagawa et al., 1993a). This isoform of MUSPC6 (MUSPC6B) ismembrane-bound, whereas the previously documented MUSPC6 is soluble (DeBie et al., 1996). Consequently, in order to avoid confusion, thepreviously documented isoform of MUSPC6 disclosed herein is designatedMUSPC6A, and the membrane-bound isoform is designated MUSPC6B. Fromcomparison of the cDNA and protein sequences, it appears that MUSPC6Aand MUSPC6B are generated via alternative splicing of the same primarytranscript (Nakagawa et al., 1993a). It appears that the 5.5 kb mRNAtranscript which we have identified in the uterus is unique to thepregnant uterus, and may encode an isoform of PC6 which is differentfrom PC6A or PC6B.

In order to confirm the presence of a unique 5.5 kb transcript in theimplantation sites in the uterus, and to determine whether this 5.5 kbtranscript is different from the 6.5 kb transcript present in theintestine and therefore may code for another isoform of MUSPC6, thefollowing approaches were used.

Firstly, cDNA fragments coding for the different domains of mouse MUSPC6protein were RT-PCR cloned, and these fragments were used as cDNA probesfor Northern blotting performed on mouse uterine tissues and theintestine. At the protein level, the MUSPC6 protein consists of thefollowing five domains (in the order from the N- to C-terminal): signalpeptide, propeptide, subtilisin-like catalytic domain, homo B domain andcysteine (Cys)-rich domain (Nakagawa et al. 1993). The first fourdomains are exactly the same between the Sciences, 20th Edition,Williams and Wilkins, Pennsylvania, USA.

The compounds and compositions of the invention may be administered byany suitable route, and the person skilled in the art will readily beable to determine the most suitable route and dose for the condition tobe treated. Dosage will be at the discretion of the attendant physicianor veterinarian, and will depend on the nature and state of thecondition to be treated, the age and general state of health of thesubject to be treated, the route of administration, and any previoustreatment which may have been administered.

The carrier or diluent, and other excipients, will depend on the routeof administration, and again the person skilled in the art will readilybe able to determine the most suitable formulation for each particularcase.

As used herein, the term “therapeutically effective amount” means anamount of a compound of the present invention effective to yield adesired therapeutic response, for example to prevent or treat a diseasewhich is susceptible to treatment by administration of apharmaceutically-active agent.

The specific “therapeutically effective amount” will of course vary withsuch factors as the particular condition being treated, the physicalcondition and clinical history of the subject, the type of animal beingtreated, the duration of the treatment, the nature of concurrent therapy(if any), and the specific formulations employed and the structure ofthe compound or its derivatives.

As used herein, a “pharmaceutical carrier” is a pharmaceuticallyacceptable solvent, suspending agent, excipient or vehicle fordelivering the compound of formula I and/or pharmaceutically-activeagent to the subject. The carrier may be liquid or solid, and is MUSPC6Aand MUSPC6B isoforms, and differences are found only in the Cys-richdomain, which is at the C-terminus. There are two unique features of theB isoform:

-   -   (1) It has a very long Cys-rich domain, which is about four        times large as that of the A isoform,    -   (2) It contains a stretch of hydrophobic amino acids as a        putative trans-membrane domain near the C-terminus (Nakagawa et        al. 1993).

Based on this protein domain information, the following cDNA fragmentswere cloned using RT-PCR:

-   -   (1) MUSPC6-fragA, 171 bp, covering nt 4-174 of MUSPC6A cDNA        (Accession: D12619; 2994 bp), representing the 5′-untranslated        region (UTR) and the signal peptide domain;    -   (2) MUSPC6-fragDA, 177 bp, covering nt 267-443 of MUSPC6A cDNA,        representing the propeptide domain;    -   (3) MUSPC6-fragDB, 479 bp, covering nt 444-922 of MUSPC6A cDNA,        representing the subtilisin-like catalytic domain;    -   (4) MUSPC6-fragG, 653 bp, covering nt 1305-1957 of MUSPC6A cDNA,        representing the Homo-B domain;    -   (5) MUSPC6-fragH, 672 bp, covering nt 2106-2777 of MUSPC6A cDNA,        representing the common Cys-rich domain present in both the A        and B isoforms;    -   (6) MUSPC6-fragI, 227 bp, covering nt 2735-2961 of MUSPC6A cDNA,        representing the C-terminus and 3′ UTR of the A isoform, and        thus representing a cDNA fragment specific to the A isoform        only;    -   (7) MUSPC6-fragK, 377 bp, covering nt 2522-2898 of MUSPC6B cDNA        (Accession: D17583, 5208 bp), representing the unique Cys-rich        domain of the B isoform;    -   (8) MUSPC6-fragM, 398 bp, covering nt 4285-4682 of MUSPC6B cDNA,        representing the unique trans-membrane domain of the B isoform.

Northern blots containing mouse uterine tissues of non-pregnant(estrus), implantation and interimplantation sites on day 4.5 and 5.5 ofpregnant mice and intestine were prepared and probed with the cDNAfragments described above. The results are shown in FIG. 6. When thefragments MUSPC6-fragA/DA/DB/G/H were used as probes, all of themultiple bands observed previously for the uterus and intestine weredetected in both type of tissues, and the 6.5 kb B-isoform specifictranscript was detected only in the intestine, as shown in FIG. 6A. Inall cases, a 5.5 kb uterus-specific transcript was indeed present in theimplantation sites, and this transcript seems to have replaced the 6.5kb transcript present in the intestine, as shown in FIG. 6A.

This indicates that the cDNA sequences tested so far are common to allof the multiple transcripts, and that therefore the protein domainsrepresented by these cDNAs are common to all of the possible isoforms ofthe MUSPC6 protein. These results also confirmed that the mRNAtranscript profiles are different between the uterus and the intestine.When MUSPC6-fragI, an A-isoform specific cDNA fragment, was used as aprobe on the same blot, only the 3.5 kb transcript, but not the othertranscripts, was detected in all of the tissues; certainly this probedid not detect the 6.5 kb transcript of the intestine, as shown in FIG.6B, indicating that only the 3.5 kb transcript but not the other onescontains this A-isoform specific cDNA sequence. This result alsoconfirms the known difference between the A and B isoforms at the cDNAlevel. When the two B-isoform specific cDNA fragments, MUSPC6-fragK andMUSPC6-fragM, were used on the same blot, in both cases the 6.5 kbtranscript was detected only in the intestine; no other bands wereobserved in either the intestine or the uterus, as shown in FIG. 6C.

When these B-isoform specific probes were tested on the multi-organNorthern blot, only the intestine showed a clear 6.5 kb band; othertissues, including muscle, brain and uterus were negative. These resultsare shown in FIG. 7, and both confirmed the specificity of the probesused in this study, and verified that the B-isoform is only expressed inthe intestine, and not in the uterus or other organs.

Collectively, our results confirm that:

-   -   (a) the 5.5 kb transcript detected in the uterus is different        from the 6.5 kb one in the intestine,    -   (b) this 5.5 kb transcript is indeed specific to the        implantation sites of the uterus during the embryo implantation        period, and    -   (c) this uterine-specific transcript may code for an isoform of        MUSPC6 which is different from the previously identified A and        from the B isoforms, the difference is mainly at the C-terminal        end.

EXAMPLE 11 Genomic Southern Analysis of MUSPC6 Gene

Genomic DNA was isolated from mouse uterus and kidney, and the DNA wassubjected to Southern analysis. Similar results were obtained for thetwo tissue types; thus only the results obtained with the uterus will bediscussed. FIG. 8 shows the results of Southern analysis of mousegenomic DNA digested separately with BamHI, EcoRI, HindIII and Bg1II andprobed with radio-labeled MUSPC6-fragH. In all cases, the digestionpattern was quite simple, and indicate that the MUSPC6 gene isrepresented by a single copy in the genome.

EXAMPLE 12 In Situ Hybridisation of MUSPC6 mRNA in the Uterus of MiceDuring Early Pregnancy

The cell types which express MUSPC6 mRNA in the uterus were identifiedby in situ hybridisation, using riboprobes generated against clone 9.5.The sequences of the riboprobes are set out in Table 2. In non-pregnantand in day 3.5 pregnant uterus, no single cell type was distinctivelypositive. Positive signals were only detected from day 4.5-6.5 ofpregnancy at the implantation sites, and they were predominantlylocalised in the decidualized stromal cells at the anti-mesometrial poleof the uterus, as shown in FIG. 9; throughout this period of time, theinterimplantation sites were always negative. Interestingly, at theimplantation sites the mesometrial side of the uterus on the samesection always had many fewer positive cells, compared to theanti-mesometrial pole. The intensity of the signals was high on day 4.5and 5.5, and then decreased dramatically on day 6.5. After day 6.5 ofpregnancy, no signals were detected in any cells in the uterus.

These results indicate that MUSPC6 mRNA is only expressed in thedecidual zones of the uterus around the embryo implantation period, andthat this expression is quite transient, and specific to the decidua.The expression is not uniformly distributed at the implantation sites;the anti-mesometrial pole, where the embryo contacts with the uterusduring implantation, showed higher expression, and the opposite side,the mesometrial pole, showed a much lower level of expression.Interestingly, some of the cells in artificially decidualized uteruswere also positive for MUSPC6 probe. This indicates that MUSPC6 isinvolved in the decidualization process, and that it may not berestricted to the embryo-induced decidualization, but also occurs inother decidualization events which can be induced by other means, suchas oil.

EXAMPLE 13 Detection of PC6 in Human Uterus and Other Human Tissues

A human PC6 cDNA probe was prepared by RT-PCR and cloned, and applied toa human multi-tissue array (Clontech). The results are illustrated inFIG. 10. A strong positive signal was detected in the heart,gastrointestinal tract, kidney, thymus, lung, placenta, uterus, testis,ovary, in similar fetal organs, and in colorectal adenocarcinoma.

A human multiorgan Northern blot with 1(g of polyA+ RNA (Clontech),applied to each lane (was probed as shown in FIG. 11. Strong positivesignals were obtained for heart, kidney, small intestine, placenta, andlung. Transcript sizes detected were around 10 kb, 6.5 kb and 3.5 kb.

FIG. 12 shows the results of semi-quantitative RT-PCR Southern blotanalysis of PC6 in human endometrium taken across the menstrual cycle.Primers used were upper primer: 5′ GAT CGG TGC ACT GAA GGA CTA 3′ (SEQID NO: 35), lower primer: 5′ CCA GCA TTC GCA CTC CTC 3′ (SEQ ID NO: 51).An expected band of 545 bp was detected in all samples.

FIG. 13 shows the results of similar semiquantitative RT-PCR analysis infirst trimester decidua and placenta, heart, pre- and post-menopausalovary and term placenta. Thus it is apparent that PC6 is expressed inthe human endometrium and a number of other human tissues. Itsexpression in decidua and placenta of early pregnancy suggests a role inhuman pregnancy.

EXAMPLE 14 Expression of PC6 in Human Endometrial Cancers

FIG. 14 shows the results of real-time quantitative RT-PCR performedusing primers on 13 samples of endometrial cancer. Messenger RNA valuesfor PC 5/6 have been corrected for loading, as assessed by quantitationfor glyceraldehyde phosphate dehydrogenase. PC6 was detected in everycancer sample, and for each grade of the cancer (1-3), InternationalFederation of Gynaecology and obstetrics Classification).

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purposes of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and areincorporated herein by this reference.

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1-29. (canceled)
 30. A nucleic acid molecule encoding an isoform of theprotein proprotein convertase (PC) 5/6, which nucleic acid molecule: (a)encodes a uterus-specific RNA transcript of about 5.5 kb; (b) isexpressed during the implantation period at implantation sites in apregnant uterus but is not detectably expressed in intestine or othertissues where PC 5/6 protein may be present; (c) under at leastmoderately stringent conditions, is able to hybridize to DNA of sequenceSEQ ID NO:4 but not to DNA of sequence SEQ ID NO:5 and SEQ ID NO:6. 31.A nucleic acid molecule according to claim 30 which is in the anti-senseorientation.
 32. A nucleic acid molecule according to claim 30, which isa cDNA molecule, a genomic DNA molecule, or an RNA molecule.
 33. Anucleic acid molecule according to claim 32, wherein the nucleic acidmolecule is a cDNA molecule.
 34. A nucleic acid molecule according toclaim 30, which: (a) comprises the sequence SEQ. ID. NO: 1 or SEQ IDNO:2; or (b) is able to hybridize under at least moderately stringentconditions to DNA having the sequence SEQ ID NO:1 or SEQ ID NO:2; or (c)has at least 75% sequence identity to SEQ ID NO:1 or SEQ ID NO:
 2. 35. Anucleic acid molecule according to claim 34, which is able to hybridizeunder stringent conditions to DNA with the sequence SEQ. ID. NO:1 or SEQID NO:
 2. 36. A nucleic acid molecule according to claim 34, which hasat least 80%, sequence identity to SEQ ID NO:1 or SEQ ID NO:2.
 37. Anisolated protein having PC 5/6 activity which is encoded by the nucleicacid molecule of claim
 30. 38. A pharmaceutical composition comprisingthe PC 5/6 protein of claim 37, together with a pharmaceuticallyacceptable carrier.
 39. A method of promoting fertility in a femalemammalian subject in need of such fertility promotion, comprising thestep of stimulating the activity of proprotein convertase (PC) 5/6 inthe uterus of a said subject.
 40. A method according to claim 39,wherein the activity is stimulated by administering a PC 5/6polypeptide, or an agonist of PC 5/6.
 41. A method according to claim39, wherein the PC 5/6 is an isoform of the form of PC 5/6 that isspecifically expressed in implantation sites of the uterus during embryoimplantation.
 42. A method according to claim 39, wherein the uterus ofthe subject is converted from a non-receptive state to a receptivestate.
 43. A method according to claim 39, wherein the stimulatingpromotes implantation of a fertilized egg in the uterus.
 44. A methodaccording to claim 39, wherein the subject suffers from afertility-related condition.
 45. A method according to claim 44, whereinthe fertility-related condition is luteal phase defect, failure ofimplantation, pre-eclampsia, early abortion, intrauterine growthrestriction, abnormal uterine bleeding, endometriosis or earlyparturition.
 46. A method according to claim 39, wherein the subject isto be implanted with one of her own fertilized eggs.
 47. A methodaccording to claim 39, wherein the subject is to be implanted with adonor's fertilized egg.
 48. A method of promoting fertility in a femalemammalian subject in need of such fertility promotion, comprising thestep of stimulating, in the uterus of a said subject, the activity of aPC 5/6 isoform that is encoded by the nucleic acid molecule of claim 19.49. A method of detecting whether a female mammalian subject is in afertile state or period, whether the uterus of said subject is in areceptive state, or whether the subject is in an early stage ofpregnancy, comprising obtaining a first biological sample from thesubject and detecting the presence or measuring the activity of PC 5/6in said sample, and, optionally, comparing the result to a second orgreater biological sample (i) obtained during a nonfertile period, or(ii) obtained at a stage of the estrous cycle when the uterus is notreceptive, or (iii) obtained from a nonpregnant subject, wherein: (a)the presence of PC 5/6, or an increase in the activity of PC 5/6 in thefirst sample relative to the activity in the second sample obtainedduring a nonfertile period, indicates that the subject is in a fertilestate or period; (b) the presence of PC 5/6 or an increased amount of PC5/6 in the first sample compared with the PC 5/6 from the second samplefrom a nonreceptive stage of the cycle indicates that the uterus is inthe receptive state; and (c) the presence of PC 5/6 activity in thefirst sample or an increase of PC 5/6 in said first sample compared tothe level in the second sample from a non-pregnant subject indicatessaid early stage of pregnancy.
 50. A method according to claim 49,wherein the biological sample is uterine tissue, washings from a uterinecavity, blood, plasma, serum or saliva.
 51. A method of inhibitingfertility in a female mammalian subject in need or desirous thereof,comprising the step of administering an antagonist of PC 5/6 to saidsubject.
 52. A method according to claim 51, wherein the antagonistinhibits conversion of a uterine non-receptive state to a receptivestate.
 53. A method according to claim 51, wherein the antagonist is ananti-PC 5/6 antibody or an anti-sense nucleic acid that specificallyhybridizes in vivo with a nucleic acid encoding PC 5/6.
 54. A methodaccording to claim 51, wherein the antagonist inhibits embryoimplantation.
 55. A method of (i) screening a sample or collection ofcompounds for a compound that modulates the enzymatic activity of PC 5/6protein, (ii) testing a candidate compound for said modulating activity,or (iii) testing a candidate compound for PC 5/6 enzymatic activity, themethod comprising: (a) providing a composition comprising said PC 5/6protein and assessing the ability of said sample or collection, or saidcandidate compound, to increase or decrease the enzymatic activity ofsaid protein relative to the activity of said PC 5/6 protein in theabsence of said sample, collection or candidate compound; or (b) testingthe candidate compound for it ability to catalyze the conversion ofprotein precursors into mature proteins, which protein precursors areones that are cleaved by PC 5/6 to become mature proteins.